e59d8f00740306de7c50bf16189e3127 pmc_34578263.pdf 9a161c4979aefec086ccfb34f454753c0f0aa4cd pmc_34578263.pdf 46338b2c6ffc3eb090ba402e6df432b434437685de885b740860455b58aef224 pmc_34578263.pdf Title: Deletion Mutants of the Attenuated Recombinant ASF Virus, BA71CD2, Show Decreased Vaccine Efficacy Subject: African swine fever (ASF) has become the major threat to the global swine industry. Lack of available commercial vaccines complicates the implementation of global control strategies. So far, only live attenuated ASF viruses (ASFV) have demonstrated solid protection efficacy at the experimental level. The implementation of molecular techniques has allowed the generation of a collection of deletion mutants lacking ASFV-specific virulence factors, some of them with promising potential as vaccine candidates against the pandemic genotype II ASFV strain currently circulating in Africa, Europe, Asia and Oceania. Despite promising results, there is room for improvement, mainly from the biosafety point of view. Aiming to improve the safety of BA71CD2, a cross-protective recombinant live attenuated virus (LAV) lacking the ASFV CD2v gene (encoding -glucuronidase as a reporter gene) available in our laboratory, three new recombinants were generated using BA71CD2 as a template: the single mutant BA71CD2f, this time containing the fluorescent mCherry reporter gene instead of CD2v, and two double recombinants lacking CD2v and either the lectin gene (EP153R) or the uridine kinase (UK) gene (DP96R). Comparative in vivo experiments using BA71CD2f, BA71CD2DP96R and BA71CD2EP153R recombinant viruses as immunogens, demonstrated that deletion of either DP96R or EP153R from BA71CD2f decreases vaccine efficacy and does not improve safety. Our results additionally confirm ASFV challenge as the only available method today to evaluate the protective efficacy of any experimental vaccine. We believe that understanding the fine equilibrium between attenuation and inducing protection in vivo deserves further study and might contribute to more rational vaccine designs in the future. Keywords: African swine fever (ASF); African swine fever virus (ASFV); live attenuated virus (LAV); vaccine; virulence factor; double mutant; protection Author: Elisabeth Lopez, Laia Bosch-Camós, Elizabeth Ramirez-Medina, Elizabeth Vuono, Maria Jesus Navas, Marta Muñoz, Francesc Accensi, Jinya Zhang, Uxia Alonso, Jordi Argilaguet, Maria Luisa Salas, Nikolay Anachkov, Douglas P. Gladue, Manuel V. Borca, Sonia Pina-Pedrero and Fernando Rodriguez Creator: LaTeX with hyperref Producer: pdfTeX-1.40.21 CreationDate: Wed Aug 25 12:33:21 2021 CEST ModDate: Wed Aug 25 12:40:19 2021 CEST Tagged: no UserProperties: no Suspects: no Form: none JavaScript: no Pages: 13 Encrypted: no Page size: 595.276 x 841.89 pts (A4) Page rot: 0 File size: 1775022 bytes Optimized: no PDF version: 1.7 name type encoding emb sub uni object ID ------------------------------------ ----------------- ---------------- --- --- --- --------- SOZKOC+VnURWPalladioL-Bold Type 1 Custom yes yes no 10 0 RAFMMA+VnURWPalladioL Type 1 Custom yes yes no 15 0 BBSDEQ+EURM10 Type 1 Builtin yes yes no 19 0 EVEJBN+URWPalladioL-Ital Type 1 Custom yes yes no 23 0 KBUBWR+URWPalladioL-Bold Type 1 Custom yes yes no 28 0 PDXFNG+PazoMath-Bold Type 1 Builtin yes yes no 32 0 EJLVIH+URWPalladioL-Roma Type 1 Custom yes yes no 36 0 MPUDFL+PazoMath Type 1 Builtin yes yes no 40 0 HOWKDX+TimesNewRomanPS-BoldItalicMT Type 1C WinAnsi yes yes no 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2023-03-31 12:05:47 CEST Size: 1775022 Format: PDF Version: 1.7 Status: Well-Formed and valid SignatureMatches: PDF-hul MIMEtype: application/pdf PDFMetadata: Objects: 708 FreeObjects: 1 IncrementalUpdates: 0 DocumentCatalog: PageLayout: SinglePage PageMode: UseNone Outlines: Item: Title: Introduction Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@44c8afef Item: Title: Methods Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@7b69c6ba Children: Item: Title: Cells and Viruses Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@46daef40 Item: Title: Generation of the Recombinant Deletion Mutants and Comparative In Vitro Growth Curves Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@12f41634 Item: Title: Animals, Hosting and Welfare Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@13c27452 Item: Title: In Vivo Experimental Approach Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@262b2c86 Item: Title: Analytical Methods Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@371a67ec Children: Item: Title: Antibody Detection by ELISA Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@5ed828d Item: Title: T-Cell Response by ELISPOT Assay Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@50d0686 Item: Title: Virus Quantification Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@7a3d45bd Item: Title: Results and Discussion Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@1e7c7811 Children: Item: Title: Replication of Recombinant Viruses in Swine Macrophage Cultures Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@77ec78b9 Item: Title: Comparative Protective Efficacy Induced by BA71CD2EP153R and BA71CD2DP96R Recombinant Viruses Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@1a3869f4 Item: Title: Evaluation of the Immune Response Induced by BA71CD2EP153R and BA71CD2DP96R Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@a38d7a3 Item: Title: References Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@77f99a05 Info: Title: Deletion Mutants of the Attenuated Recombinant ASF Virus, BA71CD2, Show Decreased Vaccine Efficacy Author: Elisabeth Lopez, Laia Bosch-Camós, Elizabeth Ramirez-Medina, Elizabeth Vuono, Maria Jesus Navas, Marta Muñoz, Francesc Accensi, Jinya Zhang, Uxia Alonso, Jordi Argilaguet, Maria Luisa Salas, Nikolay Anachkov, Douglas P. Gladue, Manuel V. Borca, Sonia Pina-Pedrero and Fernando Rodriguez Subject: African swine fever (ASF) has become the major threat to the global swine industry. Lack of available commercial vaccines complicates the implementation of global control strategies. So far, only live attenuated ASF viruses (ASFV) have demonstrated solid protection efficacy at the experimental level. The implementation of molecular techniques has allowed the generation of a collection of deletion mutants lacking ASFV-specific virulence factors, some of them with promising potential as vaccine candidates against the pandemic genotype II ASFV strain currently circulating in Africa, Europe, Asia and Oceania. Despite promising results, there is room for improvement, mainly from the biosafety point of view. Aiming to improve the safety of BA71CD2, a cross-protective recombinant live attenuated virus (LAV) lacking the ASFV CD2v gene (encoding -glucuronidase as a reporter gene) available in our laboratory, three new recombinants were generated using BA71CD2 as a template: the single mutant BA71CD2f, this time containing the fluorescent mCherry reporter gene instead of CD2v, and two double recombinants lacking CD2v and either the lectin gene (EP153R) or the uridine kinase (UK) gene (DP96R). Comparative in vivo experiments using BA71CD2f, BA71CD2DP96R and BA71CD2EP153R recombinant viruses as immunogens, demonstrated that deletion of either DP96R or EP153R from BA71CD2f decreases vaccine efficacy and does not improve safety. Our results additionally confirm ASFV challenge as the only available method today to evaluate the protective efficacy of any experimental vaccine. We believe that understanding the fine equilibrium between attenuation and inducing protection in vivo deserves further study and might contribute to more rational vaccine designs in the future. 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